Novel immunotherapy

ABSTRACT

Disclosed are treatment agents and methods of treatment utilizing the agents directed toward diseases in which the disease causing pathogen includes α6β1 integrin receptors and/or α6β4 integrin receptors on the surface of the pathogen. In one embodiment, the disease can be breast cancer. The therapeutic agents disclosed include a polypeptide comprising at least a portion of the G domain of the laminin-5 α3 chain that has been shown to bind α6β1 integrin receptors and α6β4 integrin receptors. In one embodiment, the therapeutic agents can be fused or chimeric materials in which the laminin-5 α3 chain polypeptide has been chemically bound to another material that can be useful in the destruction or neutralization of the pathogen.

BACKGROUND OF THE INVENTION

While treatment of disease has progressed far in the last century, manycurrent treatments leave much to be desired when considering the overallwelfare of the patient. For instance, currently, chemotherapy andradiation treatment remain the most widely used forms of cancertreatment known. However, such treatments are generic in their attack onthe patient's system, attacking both healthy as well as diseased tissuesand systems.

Many pathogenic agents, such as cancer cells, for example, use naturallyoccurring cellular surface receptors, primarily integrins, not only toinvade healthy tissue but also for motility within the host body, which,in the case of cancer, can lead to metastasis. Integrins are part of alarge family of cell adhesion receptors that are involved incell/extracellular matrix as well as cell/cell interaction. Integrinsare the main method that cells utilize to bind to and respond to theextracellular matrix. Functionally, integrin receptors are composed oftwo transmembrane glycoprotein subunits, an α subunit and a β subunit.Presently, 16 α and 8 β, subunits have been identified.

Pathogens which contain and utilize integrin receptors for interactionwith the extracellular matrix or cells of a host can exhibit highlyefficient invasion of and motility within an organism due to the natureof the integrin/ligand interactions. Specifically, individual integrinreceptors bind their ligands with low affinity (on the order of 10⁻⁶ to10⁻⁹ liters/mole), however, they also exist on cell surfaces in veryhigh concentration, generally 10 to 100 times greater than other typesof cell-surface receptors. Following suitable stimulation, integrins ona cell surface will cluster and form hemidesmosomes which can provide afocal contact for adhesion. The combined weak affinities of the multipleintegrins at the focal contact can give rise to a spot on the cellsurface with suitable adhesive capacity to form an adherence to theligand. This binding motif provides a method for a singleintegrin-containing cell to bind simultaneously but weakly to a largenumber of matrix molecules while still maintaining the ability toexplore the cellular environment. The low affinity integrin/ligandbinding motif thus provides an efficient route for integrin-containingpathogens to bind to and invade healthy cells while still maintainingcell motility for further invasion.

What is needed in the art are novel treatment methods for disease thatcan specifically target the pathogens of the disease. Specifically, whatis needed in the art are treatment methods that can interfere with thebinding processes of the pathogens and prevent initial invasion ofhealthy cells and motility of the pathogens within the body.Additionally, what is needed in the art is a method to specificallytarget and bind pathogens with disease fighting agents, such aschemotherapy agents, while not grossly interfering with the healthysystems and tissue that the disease has not yet affected.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention, a therapeuticcomposition for treatment of disease is disclosed. The therapeuticcomposition can include a polypeptide capable of binding to at least oneof α6 β1 integrin receptor and α6 β4 integrin receptor, wherein thepolypeptide comprises the G domain of the laminin-5 α3 chain or afragment, mutant, homolog, ortholog, analog, or allele thereof. Thetherapeutic composition also includes a pharmaceutically compatiblecarrier for the polypeptide.

In one particular embodiment, the polypeptides of the present inventioncan comprise the polypeptide as disclosed in SEQ ID NO:2, or a fragment,mutant, homolog, ortholog, analog, or allele thereof. Optionally, thepolypeptides of the present invention can comprise the polypeptide asdisclosed in SEQ ID NO:4 or SEQ ID NO: 6 or fragments, mutants,homologs, orthologs, analogs, or alleles of such. In one embodiment, thepolypeptide can have at least 70% sequence identity with the referencesequence, i.e., SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

The therapeutic composition of the present invention can be in anysuitable form. For instance, the composition can be a solid or a liquidcomposition. In various embodiments, the pharmaceutically compatiblecarrier can include a gelatin, water, an oil, or a sustained releasematrix.

The compositions of the present invention can also include othertherapeutic agents, in addition to the disclosed polypeptides. Forinstance, the compositions can include one or more chemotherapeuticagents or radioactive agents for treatment of a disease, such as acancer.

In one embodiment, the therapeutic agent of the present invention can bea fused or chimeric polypeptide agent. According to this embodiment, thetherapeutic agent can include a first component comprising a polypeptidecapable of binding to at least one of α6 β1 integrin receptor and α6 β4integrin receptor, wherein the polypeptide comprises the G domain of thelaminin-5 α3 chain or a fragment, mutant, homolog, ortholog, analog, orallele thereof. The agent can also include a second component that ischemically bound to the first component. The second component can be anyagent for use in the treatment of the disease. For instance, the secondcomponent can contribute to the destruction or neutralization of thepathogen bound by the polypeptides of the invention.

The second component can be a protein or a non-protein agent, asdesired. For example, the second component can be cytokines, wholeantibodies or fractions thereof, cell-surface receptors, ligands forcell-surface receptors, or any suitable organic molecules.

The disclosed invention is also directed to the nucleotide sequencesthat encode the therapeutic agents. In one particular embodiment, thedisclosed invention is directed to an isolated polynucleotide includinga first nucleotide sequence encoding the disclosed polypeptides and asecond nucleotide sequence that encodes a polypeptide for use in thedestruction or neutralization of the pathogens that can be bound by thepolypeptides of the invention.

In various embodiments, the first nucleotide sequence can have at leastabout 70% sequence identity with SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

The isolated polynucleotide can, in one embodiment be operably linked toan expression control sequence.

The invention is also directed to a host cell transformed with thedisclosed polynucleotides. A host cell can be, for example, a bacterial,yeast, mammalian, insect, or plant cell.

The present invention is also directed to methods of treating a disease,such as, for example, breast cancer, with the disclosed therapeuticagents. In general, the methods include contacting a pathogen thatincludes α6 β1 integrin receptors and/or α6 β4 integrin receptors on thesurface thereof with the therapeutic agents as herein described. Uponcontact, the therapeutic agents can then bind to the pathogen.

For example, the method can be carried out in vivo and the therapeuticagent can contact the pathogen via a suitable pharmaceuticallyacceptable administration system. The binding of the agent to thepathogen can mask the integrin receptors of the pathogen and thusprevent the pathogen from binding to the extracellular matrix of thehost. In certain embodiments, the method can also include the deliveryof a second component of the therapeutic agent to the pathogen, thesecond component aiding in the destruction or neutralization of thepathogen.

Pharmaceutically acceptable administration systems can include, forexample, parenteral systems, oral systems, and sustained releasesystems.

BRIEF DESCRIPTION OF THE FIGURES

A full and enabling disclosure of the present invention, including thebest mode thereof, to one of ordinary skill in the art, is set forthmore particularly in the remainder of the specification, includingreference to the accompanying figures, in which:

FIG. 1 depicts the nucleotide (SEQ ID NO: 1) and the amino acid (SEQ IDNO: 2) sequences of the complete globular domain G-1 to G-5 of theRattus norvegicus laminin-5 α3 chain;

FIG. 2 depicts the nucleotide (SEQ ID NO: 3) and the amino acid (SEQ IDNO: 4) sequences of the globular subdomains G-1 through G-3 of theRattus norvegicus laminin-5 α3 chain;

FIG. 3 depicts the nucleotide (SEQ ID NO: 5) and the amino acid (SEQ IDNO: 6) sequences of the G-3 subdomain of the Rattus norvegicuslaminin-5×3 chain;

FIG. 4 depicts a restriction map for plasmid 5C5 and plasmid 3′α3utilized in the Examples;

FIG. 5 is a table depicting cloning and expression vectors constructedand utilized in Example 1; and

FIGS. 6-9 graphically illustrate results obtained in Examples 2-5,respectively.

DEFINITIONS OF TERMS

“Polypeptide” is herein defined to indicate a molecular chain of aminoacids and does not refer to a specific length of the product. Thus,peptides, oligopeptides and proteins are included within the definitionof polypeptide. This term is also intended to include polypeptides thathave been subjected to post-expression modifications such as, forexample, glycosylations, acetylations, phosphorylations and the like.

For purposes of this disclosure, the term “protein” is herein defined toinclude any molecular chain of amino acids that is capable ofinteracting structurally, enzymatically or otherwise with otherproteins, polypeptides or any other organic or inorganic molecule.

The term “fragment” in reference to a protein or polypeptide is hereindefined as an amino acid sequence of that protein that is shorter thanthe entire protein, but comprising at least about 25 consecutive aminoacids of the full polypeptide.

For purposes of this disclosure, an “ortholog” is herein defined to be anucleotide or polypeptide sequence with similar function to a nucleotideor polypeptide sequence in an evolutionarily related species. Loci intwo species are said to be “orthologous” when they have arisen from thesame locus of their common ancestor. Orthologous polynucleotidesequences exist at loci in different species that are sufficientlysimilar to each other in their nucleotide sequences to suggest that theyoriginated from a common ancestral sequence. Orthologous sequences arisewhen a lineage splits into two species, rather than when a sequence isduplicated within a genome. Proteins that are orthologs of each otherare encoded by genes of two different species, and the genes are said tobe orthologous.

The term “mutant” is herein defined to be a polypeptide that includesany change in the amino acid sequence relative to the amino acidsequence of the reference polypeptide. Such changes can arise eitherspontaneously or by manipulations including those chemical derivativesbrought about by chemical energy (e.g., X-ray), other forms of chemicalmutagenesis, by genetic engineering, or as a result of mating or otherforms of exchange of genetic information. Mutations include, e.g., basechanges, deletions, insertions, inversions, translocations, orduplications. Mutants may or may not also comprise additional aminoacids derived from the process of cloning, e.g., amino acid residues oramino acid sequences corresponding to full or partial linker sequences.Mutants/fragments of the polypeptides of the present invention can alsobe generated by PCR cloning, or by Pseudomonas elastase digestion, asdescribed by Mariyama, M. et al. (1992, J. Biol. Chem. 267:1253-1258).

The term “homolog” is herein defined to describe two nucleotide orpolypeptide sequences that differ from each other by substitutions thatdo not effect the overall functioning of the polypeptide. For example,when considering polypeptide sequences, homologs include polypeptideshaving substitution of one amino acid at a given position in thesequence for another amino acid of the same class (e.g., amino acidsthat share characteristics of hydrophobicity, charge, pK or otherconformational or chemical properties, e.g., valine for leucine,arginine for lysine). Homologs also include polypeptides and nucleotidesequences including one or more substitutions, deletions, or insertions,located at positions of the sequence that do not alter the conformationor folding of the polypeptide to the extent that the biological activityof the polypeptide is destroyed. Examples of possible homologs includepolypeptide sequences including substitution of one non-polar(hydrophobic) residue such as isoleucine, valine, leucine or methioninefor one another; the substitution of one polar (hydrophilic) residue foranother such as between arginine and lysine, between glutamine andasparagine, or between threonine and serine; the substitution of onebasic residue such as lysine, arginine or histidine for another; thesubstitution of one acidic residue, such as aspartic acid or glutamicacid for the another; or the use of a chemically derivatized residue inplace of a non-derivatized residue, as long as the homolog polypeptidedisplays substantially similar biological activity to the referencepolypeptide, and in particular the ability to be recognized and be boundby α6 β1 and/or α6 β4 integrin receptors.

The term “analog” is herein defined to be a non-natural moleculesubstantially similar to either the entire reference protein orpolypeptide, or a fragment or allelic variant thereof, and havingsubstantially the same or superior biological activity. The term“analog” is intended to include derivatives (e.g., chemical derivatives,as defined above) of the biologically active polypeptide, as well as itsfragments, mutants, homologs, orthologs, and allelic variants, whichderivatives exhibit a qualitatively similar agonist or antagonist effectto that of the unmodified polypeptide.

The term “allele” of a polypeptide is herein defined to be a polypeptidesequence containing a naturally-occurring sequence variation relative tothe polypeptide sequence of the reference polypeptide. Similarly, anallele of a polynucleotide encoding the polypeptide is herein defined tobe a polynucleotide containing a sequence variation relative to thereference polynucleotide sequence encoding the reference polypeptide,where the allele of the polynucleotide encoding the polypeptide encodesan allelic form of the polypeptide.

“Operably linked” refers to a situation wherein the components describedare in a relationship permitting them to function in their intendedmanner. For instance, a control sequence “operably linked” to a codingsequence is ligated in such a manner that expression of the codingsequence is achieved under conditions compatible with the controlsequence. A “coding sequence” is a polynucleotide sequence which istranscribed into mRNA and translated into a polypeptide when placedunder the control of (e.g., operably linked to) appropriate regulatorysequences. The boundaries of the coding sequence are determined by atranslation start codon at the 5′-terminus and a translation stop codonat the 3′-terminus. Such boundaries can be naturally-occurring, or canbe introduced into or added to the polynucleotide sequence by methodsknown in the art. A coding sequence can include, but is not limited to,genomic DNA, mRNA, cDNA, and recombinant polynucleotide sequences.

The term “sequence identity,” as used herein, refers to the subunitsequence similarity between two polymeric molecules. For example, thesequence similarity between two polynucleotides or two polypeptides.When a subunit position in both of the two molecules is occupied by thesame monomeric subunit, then they are identical at that position. Theidentity between two sequences is a direct function of the number ofmatching or identical positions. For example, if half of the positionsin two peptide or compound sequences are identical, then the twosequences are 50% identical. The identity between two sequences is adirect function of the number of matching or identical positions. Thus,if a portion of the reference sequence is deleted in a particularpeptide, that deleted section is not counted for purposes of calculatingsequence identity. For example, when comparing a first polymer includingmonomers R₁R₂R₃R₄R₅R₆ with another polymer including monomersR₁R₂R₃R₄R₆, the two polymers have 5 out of 6 positions in common, andtherefore would be described as sharing 83.3% sequence identity.

The term “pathogen” is herein defined to include any disease causingagent. Pathogens can include disease causing agents that can infect ahost from an external source, such as bacteria, fungus, virus, and thelike, as well as pathogenic agents arising within the carrier of thedisease, including abnormal cells such as cancer cells.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to various embodiments of theinvention, one or more examples of which are set forth below. Eachembodiment is provided by way of explanation of the invention, notlimitation of the invention. In fact, it will be apparent to thoseskilled in the art that various modifications and variations may be madein the present invention without departing from the scope or spirit ofthe invention. For instance, features illustrated or described as partof one embodiment, may be used in another embodiment to yield a stillfurther embodiment. Thus, it is intended that the present inventioncover such modifications and variations as come within the scope of theappended claims and their equivalents.

The present invention is generally directed to a novel immunotherapy inwhich a protein or polypeptide agent is produced that is capable ofbinding to the surface receptors of a pathogen. Through this binding,the agent can mask the receptors of the pathogen and prevent the bindingof the pathogen to healthy host cells. In addition, through thisbinding, the agent can, in one particular embodiment, be utilized tocarry and bind other materials to the pathogen that can encourage thedestruction or neutralization of the pathogen. More specifically, thetherapeutic agents of the present invention comprise at least a portionof a laminin-5 chain that can be utilized as an immunotherapeutic agentagainst pathogens that include α6 β1 and/or α6 β4 integrin receptors onthe surface of the pathogen.

Laminin is one of a family of heterotrimer protein complexes formed fromvarious combinations of different α, β, and γ subunit chains. Lamininsin general can be found in the basement membranes of the extracellularmatrix and interact with other matrix macromolecules to contribute tocell differentiation, movement and maintenance. Laminin-5, comprising anα3 chain, a β3 chain, and a γ2 chain, is a member of the laminin familythat has been shown to function as an adhesion and migration componentfor certain cells. The terminal portion of the laminin-5 α3 chain, the Gdomain, is further subdivided into 5 sub-domains, G1, G2, G3, G4, andG5. The G subdomains of the laminin-5 α3 chain have been shown to benecessary for adherence of laminin-5 to cells which have certainreceptor integrins on their cell surface, specifically, cells containingα6 β1 and/or α6 β4 integrin receptors on the cell surface.

The present invention is generally directed to recognition andutilization of the binding characteristics of the G-domain of thelaminin-5 α3 chain. More specifically, according to the presentinvention, the entire laminin-5 α3 G-domain as well as significantportions of the laminin-5 α3 G-domain have been sequenced and expressedfor utilization in novel immunotherapies targeting pathogens containingspecific integrin receptors on the surface, specifically, thosecontaining α6 β1 and/or α6 β4 integrin receptors on the surface.

In one embodiment, therapeutic compositions comprising polypeptides ofthe disclosed portions of the G domain of the laminin-5 α3 chain havebeen developed that can be utilized to target and bind to the α6 β1and/or α6 β4 integrin receptors on the surface of a pathogen. Thepresent invention is also directed to mutants, homologs, orthologs,analogs, and/or allelic variants of the laminin-5 α3 G domains disclosedherein possessing the ability to be recognized and bound by α6 β1 and/orα6 β4 integrin receptors.

The laminin-5 of the present invention can be obtained from a variety ofsources. For example, while SEQ ID NO: 1-6 are particular to ratlaminin-5 G domains (and in particular, Rattus norvegicus), othersources of laminin-5 are encompassed by the disclosed invention. Suchsources include, but are not limited to mouse laminin-5, Mus musculuslaminin-5, artificial laminin-5 and human laminin-5.

In one embodiment, therapeutic agents can be developed comprising thatportion of the G-domain of the laminin-5 α3 chain that binds to α6 β1and/or α6 β4 integrin receptors, which is understood to exist in the G-3subdomain of the chain. For instance, in one embodiment, the therapeuticagents of the present invention can include polypeptides including allof the G1-G5 subdomains of the laminin-5 α3 chain as identified in SEQID NO: 2 as well as mutants, homologs, orthologs, analogs, and/orallelic variants of the laminin-5 α3 G1-G5 domain as identified in SEQID NO: 2.

In other embodiments, the therapeutic agents of the present inventioncan include only those sub-domains of the laminin-5 α3 chain believed tocontain the specific amino acid sequences that bind to the α6 β1 and/orα6 β4 integrin receptors of the cellular pathogens. For instance, in oneembodiment, the therapeutic agents of the present invention can includethe G1-G3 subdomains of the laminin-5 α3 chain as identified in SEQ IDNO: 4 as well as mutants, homologs, orthologs, analogs, and/or allelicvariants of the laminin-5 α3 G1-G3 subdomains as identified in SEQ IDNO: 4.

In another embodiment, the therapeutic agents of the present inventioncan include only the G3 subdomain of the laminin-5 α3 chain asidentified in SEQ ID NO: 6 as well as mutants, homologs, orthologs,analogs, and/or allelic variants of the laminin-5 α3 G3 subdomain asidentified in SEQ ID NO: 6.

Encompassed by the present invention are proteins and polypeptides thathave substantially the same amino acid sequence as the laminin-5 α3 Gdomains as herein disclosed as well as the polynucleotides that encodesuch. By the term “substantially the same” is meant both the polypeptideand the polynucleotide that encodes such that can be recognized andbound by α6 β1 and/or α6 β4 integrin receptors. For example, in oneembodiment, the nucleotide or amino acid sequence of the presentinvention can exhibit at least about 70% sequence identity with thereference sequence, at least about 80% sequence identity with thereference sequence, at least about 90% sequence identity, at least about95% sequence identity, or at least about 97% sequence identity with thereference sequence. Optionally, the polypeptide can be only that smallportion of the entire G domain that is recognized and bound by α6 β1and/or α6 β4 integrin receptors.

In addition, the presently disclosed invention is directed not only tothe disclosed polypeptides and the polynucleotides encoding such, but isalso directed to the vectors and host cells containing suchpolynucleotides. Vectors encompassed by the disclosed invention includeany molecules into which pieces of nucleic acid may be inserted orcloned that can transfer the nucleic acids carried thereby into a hostcell. In some embodiments of the present invention, vectors may alsobring about the replication and/or expression of the transferred nucleicacid pieces. An exemplary list of suitable vectors can include nucleicacid molecules derived from a plasmid, bacteriophage, or mammalian,plant or insect virus, or non-viral vectors such as ligand-nucleic acidconjugates, liposomes, or lipid-nucleic acid complexes.

In some embodiments of the present invention, the transferred nucleicacid molecule can be operatively linked to an expression controlsequence to form an expression vector capable of expressing thetransferred nucleic acid. Such transfer of nucleic acids is generallytermed transformation, and refers to the insertion of an exogenouspolynucleotide into a host cell, irrespective of the method used for theinsertion. For example, direct uptake, transduction, electroporation, orf-mating, as are generally known in the art, can be utilized. Theexogenous polynucleotide may be maintained as a non-integrated vector,for example, a plasmid, or alternatively, may be integrated into thehost genome.

The vector into which the disclosed polynucleotides can be cloned may bechosen because it functions in either a prokaryotic organism, aeukaryotic organism, or both, as desired. An exemplary list of possiblevectors include, for example, the PGEM™-T Easy vector, the pYES2 vector,the pPICZ.alpha series of vectors, the pET22b and pET28(a) vectors, andmodified pPICZ.alpha series vectors.

Following the cloning of a polynucleotide into a suitable vector, thevector can be transformed into an appropriate host cell. By “host cell”is meant a cell which has been or can be used as the recipient oftransferred nucleic acid by means of a vector. Host cells can beprokaryotic or eukaryotic, mammalian, plant, or insect, and can exist assingle cells, or as a collection, e.g., as a culture, or in a tissueculture, or in a tissue or an organism. Host cells can also be derivedfrom normal or diseased tissue from a multicellular organism, e.g., amammal. Host cell, as used herein, is intended to include not only theoriginal cell which was transformed with a nucleic acid, but alsodescendants of such a cell, which still contain the nucleic acid.

In one embodiment, isolated polynucleotides encoding the disclosedpolypeptides can additionally comprise a polynucleotide linker encodinga peptide. Such linkers are generally known to those of skill in the artand can comprise, for example, at least one additional codon encoding atleast one additional amino acid. Typically the linker comprises one toabout twenty or thirty amino acids. The polynucleotide linker can betranslated along with the disclosed polynucleotides resulting in theexpression of the disclosed polypeptides with at least one additionalamino acid residue at the amino or carboxyl terminus of the polypeptide.Importantly, the additional amino acid, or amino acids, do notcompromise the recognition and binding capability of the polypeptides byα6 β1 and/or α6 β4 integrin receptors.

In one embodiment, following insertion of the disclosed polynucleotideinto a vector, the vector can be transformed into an appropriateprokaryotic strain and the strain can be maintained under suitableculture conditions for the production of the encoded polypeptide.

In another embodiment of the present invention, a eukaryotic vector canbe utilized that comprises a modified yeast vector. According to oneparticular embodiment, a plasmid can be utilized that contains amultiple cloning site. In addition, the multiple cloning site can haveinserted thereto a His.Tag motif, as is generally known in the art.Optionally, the vector can be modified to add a restriction site, forexample an NdeI site. Such sites are well known to those of skill in theart. Proteins and polypeptides produced according to this particularembodiment can comprise a histidine tag motif (His.tag) comprising oneor more histidines, in one embodiment about 5-20 histidines. Of course,any tag should not interfere with the desired properties of the proteinsand product, namely, the ability for recognition and binding thereto byα6 β1 and/or α6 β4 integrin receptors.

In one embodiment of the present invention, the disclosed polypeptidescan be synthetically constructed amino acid sequences produced accordingto conventional methods of chemical synthesis as are generally known tothose in the art.

Pathogens that can be targeted by the disclosed therapeutic agents caninclude any pathogens that include α6 β1 and/or α6 β4 integrin receptorson the surface. For example, in some embodiments, the disclosedtherapeutic agents can be targeted toward certain cellular pathogens,including cancer cells, and specifically, breast cancer cells, prostatecancer cells, thyroid cancer cells, bladder cancer cells, colorectalcancer cells, intestinal cancer cells, squamous cell carcinomas,neuroblastomas, and fibrotic liver tissue cells.

In addition to the therapeutic agents herein disclosed, the presentinvention is also directed to methods for treatment of disease utilizingthe disclosed therapeutic agents. For example, in one embodiment, thedisclosed methods can be utilized for the destruction of primary andsecondary cancer or tumor cells by contacting and binding the agents tothe pathogenic cells and masking the integrin receptors of the cells,preventing communication between the pathogenic cells and the host andleading to the eventual death of the cancer cells.

In one particular embodiment, the present invention is directed to amethod for the inhibition or elimination of metastatic cancer or tumorcells arising from primary tumor sites. According to this embodiment,the therapeutic agents of the present invention can be directed toward atumor or cancer by use of any suitable pharmaceutically acceptablesystem and can contact the cancer cells in that targeted area. As thetargeted cancer cells include α6 β1 and/or α6 β4 integrin receptors onthe surface, the agents of the present invention can bind to thepathogens at the α6 β1 and/or α6 β4 integrin receptors. The binding ofthe therapeutic agents to the cellular pathogens can mask the receptorsof the cancer cells, as discussed above, and motility of the metastaticcell can be prevented. In addition, in certain embodiments of theinvention, this masking of the pathogen cannot only prevent the spreadof the cancer, but can also destroy the cancerous cells, for example inthose cases where the binding of the therapeutic agents to the cancercells also prevents the cancer cells from obtaining necessary nutrition.

In other embodiments of the invention, treatment with the therapeuticagents can be combined with other known treatment agents or methods todestroy or treat a disease. More specifically, the polypeptides of thedisclosed invention may be used in combination with themselves or othercompositions and procedures for the treatment of diseases. For instance,in one embodiment, the disclosed polypeptides can be combined withanother treatment agent such as a chemotherapeutic agent. For example, atherapeutic composition of the present invention can include thedisclosed polypeptides and a second therapeutic agent such as Vasostatinor anti-alpha 6 integrin monoclonal antibodies with a pharmaceuticallycompatible carrier. In another embodiment, a disease may be treatedconventionally with surgery, radiation, or chemotherapy, and thedisclosed polypeptides may additionally be administered to the patientto further treat the disease such as by extension of the dormancy ofmicrometastases or to stabilize and inhibit the growth of any residualprimary tumor.

In another embodiment, the disclosed polypeptides can be combined withother pharmaceutically acceptable excipients in forming therapeuticcompositions. The compositions of the present invention may additionallycontain other polypeptides or chemical compounds for disease treatmentas are generally known in the art. Such additional factors and/or agentsmay be included in the composition to produce a synergistic effect withthe polypeptides of the invention.

In one embodiment of the present invention, the disclosed polypeptidescan be chemically combined with secondary materials so as to form asingle agent comprising both materials. Generally, the second componentof the combination agent can be useful in fighting the disease, forexample can aid in destruction or neutralization of the pathogen. Forexample, in one embodiment, the recognition and binding of thepolypeptides to the pathogen can be utilized as a method for deliveringthe second component directly to the pathogen. For example, thetherapeutic agents of the present invention can include fusionpolypeptides or chimeric polypeptides comprising the disclosedpolypeptides of the laminin-5 α3 G domain, or their fragments, mutants,homologs, orthologs, analogs, and allelic variants, chemically combinedwith a secondary polypeptide material so as to form a single therapeuticagent. Exemplary secondary polypeptide materials can include, forexample, IL-2, IL-3 IL-15, IL-12, IFN-γ, GM-CSF, CD40, CD40 ligand(CD40L), C3 Complement components, CD80, CD86, FAS, or FAS ligand(FASL). The nucleotide and amino acid sequences of each of theseexemplary components are known in the art and can be found in the NBCIGenBank database.

In one embodiment, a fusion or chimeric product or polypeptide of thepresent invention can be produced as a result of recombinant expressionand the cloning process as described above, and the polypeptide may beproduced comprising additional amino acids or amino acid sequencescorresponding to full or partial linker sequences. Alternatively, afusion or chimeric product of the present invention can be a multimer ofa single polypeptide. That is, a polypeptide including one or morerepeating sequences of the disclosed polypeptides. In yet anotherembodiment, the therapeutic agents of the present invention can befusion and chimeric polypeptides that can be formed of one or more ofthe different polypeptides as herein disclosed. For example, in oneembodiment, a therapeutic agent according to the present invention caninclude a polypeptide comprising a polypeptide as disclosed according toSEQ ID NO: 2 in combination with one or more polypeptides as disclosedaccording to SEQ ID NO: 4 and/or SEQ ID NO: 6.

In yet another embodiment of the present invention, the therapeuticagent can be a fusion or chimeric product in which polypeptides asherein disclosed are chemically combined with other, non-proteinsecondary components so as to form a single therapeutic agent. Forexample, the disclosed polypeptides can be combined with additionalcomponents such as, for example, superantigens, muramyl dipeptide (MDP),lipopolysaccharide (LPS), or mannose. According to this embodiment,fusion or chimeric therapeutic agents encompassed by the presentinvention can generally include one or more of the disclosedlaminin-5×3α G domain polypeptides linked together with other materialsvia post-translational modification through covalent bonds such asamide, ester, disulfide or azo bonds, for example.

In general, methods for treatment of disease utilizing the disclosedagents include contacting the cellular pathogen with a compositioncomprising the polypeptides of the invention. For example, in oneembodiment, the methods of the disclosed invention can be utilized invivo for treatment of a disease such as cancer. According to thisembodiment, a composition including the disclosed therapeutic agents anda pharmaceutically compatible carrier can be delivered to a patient viaany pharmaceutically acceptable delivery system. For instance, acomposition of the present invention including a pharmaceuticallycompatible carrier and the disclosed polypeptides may be a solid, liquidor aerosol and may be administered by any known pharmaceuticallyacceptable route of administration. A non-limiting exemplary listing ofpossible solid compositions can include pills, creams, and implantabledosage units. An implantable dosage unit can, in one embodiment, beadministered locally, for example at a tumor site, or can be implantedfor systemic release of the composition, for example subcutaneously. Anon-limiting exemplary listing of possible liquid compositions caninclude formulations adapted for injection subcutaneously,intravenously, intraarterially, and formulations for topical andintraocular administration. Possible examples of aerosol formulationsinclude inhaler formulations for direct administration to the lungs.

The proteins and protein fragments of the disclosed invention can beprovided as isolated and substantially purified proteins and proteinfragments in pharmaceutically acceptable formulations using formulationmethods known to those of ordinary skill in the art. These formulationscan generally be administered by standard routes. For example, thecombinations may be administered by topical, transdermal,intraperitoneal, intracranial, intracerebroventricular, intracerebral,intravaginal, intrauterine, oral, rectal or parenteral (e.g.,intravenous, intraspinal, subcutaneous or intramuscular) route. Osmoticminipumps may also be used to provide controlled delivery of highconcentrations of the disclosed polypeptides through cannulae to thesite of interest, such as directly into a metastatic growth.

Pharmaceutical compositions for parenteral injection according to thepresent invention include pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyois (e.g., glycerol, propylene glycol, polyethylene glycoland the like), carboxymethylcellulose and suitable mixtures thereof,vegetable oils (e.g., olive oil) and injectable organic esters such asethyl oleate. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents and the like which enhance the effectiveness of theactive ingredient. Proper fluidity may be maintained, for example, bythe use of coating materials such as lecithin, by the maintenance of therequired particle size in the case of dispersions and by the use ofsurfactants. These compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like.

Prolonged absorption of an injectable pharmaceutical form may be broughtabout by the inclusion of agents, such as aluminum monostearate andgelatin, which can delay absorption. For example, injectable depot formscan be made by forming microencapsule matrices of the therapeutic agentin biodegradable polymers such as polylactide-polyglycolide,poly(orthoesters) and poly(anhydrides). Depending upon the ratio oftherapeutic agent to polymer and the nature of the particular polymeremployed, the rate of drug release can be controlled. Depot injectableformulations can also be prepared by entrapping the therapeutic agentsin liposomes or microemulsions which are compatible with body tissues.The injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedia just prior to use.

In one embodiment, the therapeutic compositions of the present inventioncan include pharmaceutically acceptable salts of the components therein,e.g., those that may be derived from inorganic or organic acids.Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describes pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences (1977) 66:1 et seq., which isincorporated herein by reference. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with the free amino groups ofthe polypeptide) that are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, tartaric, mandelic and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like. The salts may be prepared insitu during the final isolation and purification of the compounds of theinvention or separately by reacting a free base function with a suitableorganic acid. Representative acid addition salts include, but are notlimited to acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,digluconate, glycerophosphate, hemisulfate, heptonoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxymethanesulfonate (isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups can be quaternized with such agents as loweralkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl,and diamyl sulfates; long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides likebenzyl and phenethyl bromides and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which maybe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid.

In one embodiment the treatment method can include use of timed releaseor sustained release delivery systems as are generally known in the art.Such systems can be desirable, for instance, in situations where surgeryis difficult or impossible, e.g., situations involving patientsdebilitated by age or the disease course itself, or where therisk-benefit analysis dictates control over cure. According to thisparticular embodiment, a sustained-release matrix can include a matrixmade of materials, usually polymers, which are degradable by enzymaticor acid/base hydrolysis or by dissolution. Once inserted into the body,such a matrix can be acted upon by enzymes and body fluids. Thesustained-release matrix desirably is chosen from biocompatiblematerials such as liposomes, polylactides (polylactic acid),polyglycolide (polymer of glycolic acid), polylactide co-glycolide(co-polymers of lactic acid and glycolic acid) polyanhydrides,poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitinsulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone and silicone. Possible biodegradable polymers andtheir use are described, for example, in detail in Brem et al. (1991, J.Neurosurg. 74:441-6), which is hereby incorporated by reference in itsentirety.

When an effective amount of therapeutic agents of the present inventionis administered orally, the therapeutic compositions can be in the formof a tablet, capsule, powder, solution or elixir. When administered intablet form, the pharmaceutical composition of the invention mayadditionally contain a solid carrier such as a gelatin or an adjuvant.The tablet, capsule, or powder can, for example, contain from about 5 to95% therapeutic agents of the present invention. In one embodiment, thecomposition can contain from about 25 to 90% therapeutic agents of thepresent invention.

When administered orally in liquid form, a liquid carrier such as water,petroleum, oils of animal or plant origin such as peanut oil, mineraloil, soybean oil, or sesame oil, or synthetic oils may be added. Theliquid form of the pharmaceutical composition may further containphysiological saline solution, dextrose or other saccharide solution, orglycols such as ethylene glycol, propylene glycol or polyethyleneglycol. When administered in liquid form, the pharmaceutical compositioncontains from about 0.5 to 90% by weight of therapeutic agents of thepresent invention, in one embodiment from about 1 to 50% therapeuticagents of the present invention.

When an effective amount of the agents of the present invention areadministered by intravenous, cutaneous or subcutaneous injection, thepolypeptides of the present invention can generally be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable polypeptide solutions, having due regardto pH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection can contain, in addition to the polypeptidesof the present invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art.

The dosage of the disclosed polypeptides of the present invention candepend on the disease state or condition being treated and otherclinical factors such as weight and condition of the human or animal andthe route of administration of the compound. Depending upon thehalf-life of the disclosed polypeptides in the particular animal orhuman, the disclosed polypeptides can be administered between severaltimes per day to once a week. It is to be understood that the presentinvention has application for both human and veterinary use. The methodsof the present invention contemplate single as well as multipleadministrations, given either simultaneously or over an extended periodof time. In addition, the disclosed polypeptides can be administered inconjunction with other forms of therapy, e.g., chemotherapy,radiotherapy, or other immunotherapy.

The present invention may be better understood with respect to thefollowing examples.

EXAMPLE 1

General Methodologies for Construction, Expression, and Isolation ofLaminin-5-α3 Peptides

Manipulations of DNA were completed according to standard techniques asdescribed by Sambrook, et al., Molecular Cloning: A Laboratorv Manual,second ed., Cold Spring Harbor Laboratory Press, New York, 1989.Restriction enzymes were purchased from either Promega or New EnglandBiolabs. Escherichia coli (E. coli) strains DH5αMCR (Jessee and Bloom,1988), GM2163 (New England Biolabs), and JM109 (Promega) were usedthroughout and grown in either L. broth (Lennox, 1955) or Terrific Broth(Tartof and Hobbs, 1987). All cultures were grown at 37° C. and liquidcultures were agitated at 250 rpm. Plasmid ligation reactions wereperformed according to protocols found in Sambrook et al. (1989). Pasmidligations were transformed into E. coli competent cells (from strainsDH5αMCR or GM2163) by a heat-shock procedure (Henson, 1984) whiletransformations into E. coli competent cells from strain JM109 wereconducted according to the manufacturer's protocol. Plasmid DNA fromputative transformants was isolated using a plasmid miniprep system.

Plasmid Construction and Transformation

Two partial cDNA clones from rat laminin-5-α3 cloned into pBluescript SK(Stratagene) were obtained from Northwestern University. Plasmid 5C5encoded the region of 932-3242 bp of laminin-5 α3 chain region, and,plasmid 3′α3 encoded the 3′ half of laminin-5-α3 chain region from3092-5250 bp. FIG. 4 illustrates the restriction map.

Plasmid pHB1 was constructed from PCR amplification of the 5C5 cDNA (seeFIG. 5). For cloning in the vector pGEM-T Easy, the following syntheticoligonucleotide primers were used for PCR amplification: forward(5′-AATTAACCCTCACTAAAGGG-3′) (SEQ ID NO: 7) and reverse(5′-TAATACGACTCACTATAGGG-3′) (SEQ ID NO: 8). The addition of Taqpolymerase resulted in poly A-overhangs that were added to the PCRproducts and purified from 1% agarose gel using the Rapid PlasmidMiniprep System (Marligen Biosciences, Inc). The agarose purifiedproduct was ligated into pGEM-T Easy. The ligation reaction was carriedout for 4 hours at room temperature. E. coli strain GM2163 (New EnglandBiolabs) was transformed (Sambrook, et al 1989) with the ligationproduct. Proper construction of the final plasmid pHB1 was verified byrestriction analysis and DNA sequencing on a Licor 4200L sequencer usingT7 and SP6 primers.

Plasmid pHB2 was constructed from PCR amplification of the 3′α3 cDNA.For cloning in the vector PGEM-T Easy, the following syntheticoligonucleotides primers were used for PCR amplification: forward(5′-CCAGACTACTGTGGACAGAGG-3′) (SEQ ID NO: 9) and reverse(5′-AAGGGTTCTTCGTGTGTAGGG-3′) (SEQ ID NO: 10). Procedures for plasmidconstruction, transformation and verification were continued asdescribed previously for pHB1.

Plasmid pHB3 was constructed from pHB2 by digesting with XbaI,precipitation, followed by a partial digest with EcoRI. Aliquots wereremoved in 10 μl volumes every 3 minutes followed by the addition of 1μl 0.5 M EDTA pH 8.0. Restriction digest products were viewed on 1.5%agarose gel, and the desired 1761 bp fragment was agarose purified asdescribed above. Cloning vector pYES2 was digested by XbaI and EcoRI andligated with the 1761 bp XbaI/EcoRI partial fragment from pHB2. Theligation reaction was carried out for 24 hours at room temperature andtransformed into E. coli strain DH5αMCR. Proper construction wasverified by restriction enzyme analysis.

Plasmid pHB4 (containing G1-G5 domains of laminin-5-α3) was constructedfrom pHB1 digest with SacI/EcoRI. The resulting 822 bp fragment from 5C5was agarose purified. Plasmid pHB3 was digested with SacI, precipitated,followed by an EcoRI partial digest performed as illustrated previously.Restriction digest products were viewed on a 0.7% agarose gel and thedesired fragment containing pYES2+1761 bp was agarose purified. The 822bp fragment from pHB1 was ligated with PYES+1761 bp (linearized pHB3)for 24 hours at room temperature and transformed in E. coli strainDH5αMCR. Proper construction was verified by restriction enzymeanalysis. Internal junctions were sequenced on ABI Prism 3700 DNAAnalyzer using the primers: forward (5′-CTACTCAACCAAATGCTCCC-3′) (SEQ IDNO: 11) and reverse (5′-GTACTATTCAACCTGACAACCC-3′) (SEQ ID NO: 12).Prior to sequencing, dye-terminator removal was completed using QiagenDyeEX™ 2.0 Spin Kit.

Expression Vector Construction and Transformation in E. Coli

The expression of recombinant laminin proteins was produced using theEasySelect™ Pichia Expression Kit from Invitrogen. Cloning oflaminin-5-α3 G domains into the P. pastoris expression vector pPICZαBfor the construction of pHB6 was performed in a multi-step procedure.pPICZαB was digested with KpnI, followed by a T4 DNA polymerase reactionto blunt 3′ overhangs, precipitated, digested with XbaI and theresulting fragment was purified from a 1% agarose gel. The DNA fragmentencoding the regions of G1-G5 of laminin-5-α3 chain from pHB4 wasdigested with SacI, followed by treatment with T4 DNA polymerase,digested with XbaI and also purified from a 1% agarose gel. The ligationreaction was 24 hours at room temperature, followed by transformation inE. coli strain JM109 and plated onto low salt LB Zeocin™ plates. Properconstruction of the vector pHB6 was verified by restriction analysis andDNA sequencing. The following synthetic oligonucleotides were used: (SEQID NO: 13) forward AOX1 (5′-GACTGGTTCCAATTGACAAGC-3′) (SEQ ID NO: 14)and reverse AOX1 (5′-GCAAATGGCATTCTGACATCC-3′).

Construction of pHB7 was performed from P. pastoris cloning/expressionvector pPICZαB, and the DNA fragment encoding the regions of G3-G5 frompHB6 digestion with EcoRI/XbaI and agarose purified. The ligationreaction, transformation, restriction analysis and DNA sequencing wereall conducted as stated above for pHB6.

Production of pHB8 containing laminin-5-α3 plasmid G1-G3 domains inpPICZαB was by removal of the G4 and G5 domain from the plasmid pHB6.This was performed by digestion of pHB6 with XbaI enzyme, treated withT4 DNA polymerase to blunt the 3′ overhangs, precipitated, followed by apartial digest with PvuII and agarose purification of a 5304 bpfragment. This linear fragment was religated for 24 hours at roomtemperature and transformed into E. coli strain JM109. Construction ofplasmid pHB8 was verified by restriction analysis and DNA sequencing asstated for pHB6.

Construction of plasmid pHB9 included the G3 domain of laminin-5-α3chain cloned into the expression vector pPICZαB by the following:digestion of the vector with XbaI, treated with T4 DNA polymerase toblunt 3′ overhangs, precipitated, digested with EcoRI, and agarosepurified. The G3 insert was produced from plasmid pHB7 digested withEcoRI, precipitated, digested with PvuII and agarose purified. Ligationconditions were 24 hours at room temperature and transformed into E.coli strain JM109. Verification of correct construction was as statedfor pHB6.

Expression in P. pastoris

The P. pastoris yeast strain SMD1168 was transformed by the PichiaEasyComp™ Kit as described from Invitrogen with 5 μg of SacI linearizedexpression plasmids. Multicopy recombinants were selected on YPDS (yeastextract with peptone, dextrose and sorbitol) plates containing 100 μg/mlZeocin™. Loss of the AOX1 gene results in a strain that is referred toas Mut^(s), which is designated to describe the phenotype of suchmutants that lack the ability to metabolize methanol. Cells described asthe Mut⁺ phenotype are capable of utilizing methanol as the sole carbonsource. These phenotypes are commonly used to evaluate the P. pastoristransformants for correct integration into the genome. Transformantswere screened for their ability to grow on histidine-deficient minimaldextrose agar plates which confirmed the Mut⁺ phenotype.

Analysis of Recombinant Laminin

For verification of gene integration into the P. pastoris genome,genomic DNA was isolated and analyzed by PCR. Single colonies from theYPDS-Zeocin™ plates were used to inoculate 5 ml overnight cultures inYPD (1% yeast extract, 2% peptone, 2% dextrose) medium. The isolation ofgenomic DNA was performed by the Rather Rapid Genomic Prep protocol(Hoffman and Winston, Rather Rapid Genomic Prep, Gene, Vol. 87:262-272,1987). Direct PCR screening of P. pastoris clones was carried out usingthe synthetic oligonucleotides encoding the 5′ AOX1 (942 bp fragmentcontaining the AOX1 promoter that allows methanol-inducible, high levelexpression in P. pastoris) and 3′ AOX1 regions using the same primersdescribed previously in the expression vector construction.

High-Cell Density Expression of Recombinant P. pastoris Strains

Expression of the recombinant laminin protein was carried out in aBioFlo 110 Modular Benchtop Fermentor (New Brunswick Scientific) with atotal volume of 2.0 L. The dissolved oxygen was kept at 30% ofsaturation until feed phase, this was maintained at >40%. The dissolvedoxygen was controlled by the agitation rate. Total inlet gas flow waskept>2 vvm (v=volume of air in ml, v=per unit of medium in L, m=per unitof time in hours). The pH was maintained at 5.0 by the addition of NH₄OHwhich also served as a minor nitrogen source. The temperature wasconstant during the fermentation at 30° C.

The innoculum was prepared from 1 ml pre-cultures stored at −80° C. inglycerol with an OD₆₀₀ of 35.0. This was resuspended in 5 ml of 1% BMGYmedium and incubated at 30° C. for 4 hours at 250 rpm. Media containing1% BMGY includes 1% glycerol, 100 mM potassium phosphate, pH 6.0,1.34%YNB (yeast nitrogen base) and 4×10⁻⁵% biotin. The culture was used toinoculate 100 ml of overnight medium for an initial OD₆₀₀ of 0.5.Overnight medium was 1% BMGY with the addition of 100 μg/ml of Zeocin™.Incubation continued using the same conditions as before for 11 hourgrowth. The batch phase was the initial phase in the BioFlo fermentorusing 900 ml of batch medium with the addition of the 100 ml innoculum.Batch medium (1 L) consisted of 12 g glycerol, 100 ml 10×YNB (13.4%yeast nitrogen base with ammonium sulfate without amino acids), 100 ml 1M potassium phosphate, pH 6.0, 2 ml 500× biotin, 1 ml 1000× tracemetals, and 786 ml milliQ water. The batch phase lasted 20 hours. Duringthe glycerol fed-batch phase over the next 18 hours, a continuous feedof 444 g glycerol, 100 ml 10× YNB, 1 ml 1000× trace metals, 2 ml 500×biotin and antifoam were added. The total volume of medium added was 474ml. The methanol fed-batch phase (induction) continued for 20 hours witha medium volume of 203 ml added. This induction medium was comprised of200 ml 100% MeOH, 1 ml 1000× trace metals and 2 ml 500× biotin. Tracemetal components for all media above contained the following per liter:2.0 g CuSO₄, 0.1 g KI, 3.0 g MnSO₄.H₂O, 0.2 g Na₂MoO₄.2H₂O, 0.02 g boricacid, 0.5 g COCl₂, 7.0 g ZnCl, 10.0 g FeSO₄.H2O.

Purification of Recombinant Laminin

The supernatant containing recombinant laminin-5 G domains was collectedby centrifugation at 5000× g for 10 min. Purification chromatography wasperformed using 10 g of Sephadex™ G-75 superfine (20-50 micron particlediameter) suspended in 500 ml of 0.1 M ammonium acetate pH 6.95. Thesuspension was allowed to equilibrate overnight. The swelled gel wasdegassed for 4 hours and packed into a 1 cm×40 cm Pharmacia columnequipped with plastic frit, 10μ filter disk and flow valve. The gel wasallowed to settle at full flow rate under gravity. The column was packedto a height of 28 cm with the G-75. Void volume was determined using 3ml of 2 mg/ml blue dextran (MW>2×10⁶ Daltons) in PBS containing 1%glycerol. The sample was applied by underlayering and the column waseluded with 0.1 M ammonium acetate until the blue dextran began toemerge (25 ml) and continued until all of the blue dextran had washedfrom the column (31 ml total). The sample containing 2.5 mg/ml ofprotein in a 2 ml supernatant plus 1 ml of ammonium acetate was appliedto the column as given above. The void volume was obtained as abovefollowed by an additional total column volume of 85 ml that wascollected containing the desired recombinant protein of laminin-5. Thissample was freeze dried, resuspended in 1 ml total volume of ddH₂O andused for functional assays.

EXAMPLE 2

Adhesion characteristics of MDA-MB-435 breast cancer cells torecombinant (r) rat laminin-5 α3 chain G3 domain protein (SEQ ID NO:6)were examined.

Initially, untreated 96-well plates were coated overnight at 4° C. withpurified recombinant rat laminin-5 α3 chain G3 domain protein (SEQ IDNO:6). Various concentrations of the G3 domain protein were added totriplicate wells. Specifically, concentrations examined included 0.1,0.5, 1.0, 2.5, 5.0, 7.5 and 10 μg/ml diluted in sterile PBS. Followingovernight coating, wells were washed twice with PBS and blocked for 1hour at room temperature with 1% BSA/PSA.

MDA-MB-435 breast cancer cells were collected by brief trypsinization,washed twice with culture medium, and plated in the prepared wellsincluding control wells that had not been coated with the G3 domainprotein, at 5×10⁵ cells/well. Plates were incubated for 1 hour in a 37°C. humidified incubator (5% CO₂). After incubation, wells were washedwith medium twice to remove unbound cells, followed by fixing attachedcells with 3.7% paraformaldehyde/PBS for 10 minutes, and then stainingattached cells with 0.5% crystal violet solution. At the end of 10minutes staining, wells were washed twice with ddH₂O to remove excessdye followed by addition of 1% SDS to solubilize cells. The amount ofcrystal violet incorporated into attached cells was determined using aMolecular Devices plate reader set to absorb at 550 nm.

FIG. 6 graphically illustrates the results. Mean O.D. readings labeledon the graph with different letters are significantly different atP≦0.0001.

EXAMPLE 3

Well plates were coated overnight at 4° C. with purified recombinant ratlaminin-5 α3 chain G3 domain protein (SEQ ID NO:6) as described abovewith all coated wells coated at 5.0 μg/ml diluted in sterile PBS.Following overnight coating, wells were washed twice with PBS andblocked for 1 hour at room temperature with 1% BSA/PSA.

MDA-MB-435 breast cancer cells were collected by brief trypsinizationand washed twice with culture medium. The MDA-MB-435 cells were splitinto three portions, one portion was plated as described above inExample 2 at 5×10⁵ cells/well in wells previously coated with 5.0 μg/mlG3 domain protein (labeled G3 on FIG. 7) and also plated into otherwiseuntreated well plates (labeled Control on FIG. 7). The other twoportions were incubated prior to plating with either anti-α6 integrinmonoclonal antibody or mouse IgG2a isotope control at a 1:5 dilution for15 minutes. Following incubation, these portions were also plated inwells previously coated with 5 μg/ml of the G3 protein according to theprocess described above in Example 2 at 5×10⁵ cells/well (labeledG3+Anti-alph6 and G3+IgG2a, respectively, on FIG. 7) and also wereplated in untreated well plates, i.e., wells not previously coated withthe G3 protein (labeled Anti-alpha6 and IgG2a on FIG. 7, respectively).

All plates were incubated for 1 hour in a 37° C. humidified incubator(5% CO₂). After incubation, wells were washed with medium twice toremove unbound cells, followed by fixing attached cells with 3.7%paraformaldehyde/PBS for 10 minutes, and then staining attached cellswith 0.5% crystal violet solution. At the end of 10 minutes staining,wells were washed twice with ddH₂O to remove excess dye followed byaddition of 1% SDS to solubilize cells. The amount of crystal violetincorporated into attached cells was determined using a MolecularDevices plate reader set to absorb at 550 nm.

FIG. 7 graphically illustrates the results. Mean O.D. readings labeledon the graph with different letters are significantly different atP≦0.0001.

EXAMPLE 4

Purified recombinant rat laminin-5 α3 chain G3 domain protein (SEQ IDNO:6) was added in increasing concentrations to wells of 96-well platesas described above. Specifically, the concentrations of G3 domainprotein added to wells were 1.0, 2.5, 5.0, 7.5, 10 and 15 μg/ml dilutedin culture medium.

MDA-MB-435 cells were collected by brief trypsinization, washed twicewith culture medium, and plated at 5×10⁵ cells/well in the preparedwells as well as in control wells not coated with the G3 domain protein.Plates were incubated for 24 hours at 37° C. in a humidified incubator(5% CO₂). Twenty μl of MTT(3-[4,5-dimethlythiozol-2-yl]-2,5-diphenyltetrazolium bromide) reagent(5 mg/ml) was added to all wells during the final 4 hours of incubation.One hundred μl of culture supernatant was removed from each wellfollowed by the addition of 100 μl dimethylsulfoxide (DMSO) tosolubilize the cells. Plates were shaken for 15 minutes and absorbancewas recorded at dual wavelengths of 570/650 nm using a Molecular Devicesplate reader to examine the proliferation of the cancer cells in thewells.

FIG. 8 illustrates the results. Mean O.D. readings labeled on the graphwith different letters are significantly different at P≦0.0001. As canbe seen, cancer cell proliferation declined with increasing dosage levelof the G3 domain protein composition.

EXAMPLE 5

Well plates were coated overnight at 4° C. with purified recombinant ratlaminin-5 α3 chain G3 domain protein (SEQ ID NO:6) as described abovewith all coated wells coated at 10.0 μg/ml diluted in sterile PBS.Following overnight coating, wells were washed twice with PBS andblocked for 1 hour at room temperature with 1% BSA/PSA.

MDA-MB-435 breast cancer cells were collected by brief trypsinizationand washed twice with culture medium. The MDA-MB-435 cells were splitinto three portions, one portion was plated as described above inExample 4 at 5×10⁵ cells/well in wells previously coated with 10.0 μg/mlG3 domain protein (labeled G3 on FIG. 9) and also plated into otherwiseuntreated well plates (labeled Control on FIG. 9). The other twoportions were incubated prior to plating with either anti-α6 integrinmonoclonal antibody or mouse IgG2a isotope control at a 1:10 dilutionfor 15 minutes. Following incubation, these portions were plated inwells previously coated with 10 μg/ml of the G3 protein according to theprocess described above in Example 2 at 5×10⁵ cells/well (labeledG3+Anti-alph6 and G3+IgG2a, respectively, on FIG. 9) and also wereplated in untreated well plates, i.e., wells not previously coated withthe G3 protein (labeled Anti-alpha6 and IgG2a on FIG. 9, respectively).

Plates were incubated for 24 hours at 37° C. in a humidified incubator(5% CO₂). Twenty μl of MTT(3-[4,5-dimethlythiozol-2-yl]-2,5-diphenyltetrazolium bromide) reagent(5 mg/ml) was added to all wells during the final 4 hours of incubation.One hundred μl of culture supernatant was removed from each wellfollowed by the addition of 100 μl dimethylsulfoxide (DMSO) tosolubilize the cells. Plates were shaken for 15 minutes and absorbancewas recorded at dual wavelengths of 570/650 nm using a Molecular Devicesplate reader to examine the proliferation of the cancer cells in thewells.

FIG. 9 illustrates proliferation results. Mean O.D. readings labeled onthe graph with different letters are significantly different atP≦0.0001. As can be seen, proliferation declined with the G3 domainprotein and with the Anti-x 6 antibody, with the combination of the twoexhibiting the best results.

It will be appreciated that the foregoing examples, given for purposesof illustration, are not to be construed as limiting the scope of thisinvention. Although only a few exemplary embodiments of this inventionhave been described in detail above, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention which isdefined in the following claims and all equivalents thereto. Further, itis identified that many embodiments may be conceived that do not achieveall of the advantages of some embodiments, yet the absence of aparticular advantage shall not be construed to necessarily mean thatsuch an embodiment is outside the scope of the present invention.

1-14. (canceled)
 15. A therapeutic agent comprising: a fused or chimericpolypeptide comprising a first component comprising a polypeptide thatspecifically binds to at least one of α6β1 integrin receptor and α6β4integrin receptor, wherein the polypeptide comprises the G3 subdomain ofthe laminin-5 α3 chain or a fragment, mutant, homolog, ortholog, analog,or allele thereof, and a second component chemically bound to said firstcomponent, wherein said second component comprises an agent for use inthe destruction or neutralization of a pathogen comprising at least oneof α6 β1 integrin receptors and α6 β4 integrin receptors on the surfaceof the pathogen.
 16. The therapeutic agent of claim 15, wherein thesecond component is a polypeptide.
 17. The therapeutic agent of claim15, wherein the second component is a non-protein agent.
 18. Thetherapeutic agent of claim 15, wherein the second component is selectedfrom the group consisting of IL-2, IL-3 IL-15, IL-12, IFN-γ, GM-CSF,CD40, CD40 ligand (CD40L), C3 Complement components, CD80, CD86, FAS,FAS ligand (FASL), superantigens, muramyl dipeptide (MDP),lipopolysaccharide (LPS), or mannose
 19. The therapeutic composition ofclaim 15, wherein the first component comprises at least about 70%sequence identity with SEQ ID NO:2.
 20. The therapeutic composition ofclaim 15, wherein the first component comprises at least about 70%sequence identity with SEQ ID NO:4.
 21. The therapeutic composition ofclaim 15, wherein the first component comprises at least about 70%sequence identity with SEQ ID NO:6. 22-42. (canceled)
 43. Thetherapeutic composition of claim 15, wherein the first componentcomprises at least about 90% sequence identity with SEQ ID NO:2.
 44. Thetherapeutic composition of claim 15, wherein the first componentcomprises at least about 90% sequence identity with SEQ ID NO:4.
 45. Thetherapeutic composition of claim 15, wherein the first componentcomprises at least about 90% sequence identity with SEQ ID NO:6.
 46. Thetherapeutic composition of claim 15, wherein the first componentconsists of SEQ ID NO:6.
 47. The therapeutic composition of claim 15,wherein the first component comprises a segment consisting of SEQ IDNO:6.
 48. A fused or chimeric polypeptide comprising a first componentcomprising a polypeptide, wherein the polypeptide comprises at least asegment of the G-domain of a laminin-5 α3 chain and the polypeptidespecifically binds to at least one of α6 β1 integrin receptor and α6 β4integrin receptor that specifically binds to a polypeptide comprising asegment consisting of SEQ ID NO:2, and a second component chemicallybound to said first component.
 49. The fused or chimeric polypeptide ofclaim 48, wherein the second component is a polypeptide.
 50. Thetherapeutic agent of claim 48 wherein the second component is anon-protein agent.
 51. A fused or chimeric polypeptide comprising afirst component comprising a polypeptide, wherein the polypeptidecomprises at least a segment of the G-domain of a laminin-5 α3 chain andthe polypeptide specifically binds to at least one of α6 β1 integrinreceptor and α6 β4 integrin receptor that specifically binds to apolypeptide comprising a segment consisting of SEQ ID NO:4, and a secondcomponent chemically bound to said first component.
 52. The fused orchimeric polypeptide of claim 51, wherein the second component is apolypeptide.
 53. The therapeutic agent of claim 51 wherein the secondcomponent is a non-protein agent.
 54. A fused or chimeric polypeptidecomprising a first component comprising a polypeptide, wherein thepolypeptide comprises at least a segment of the G-domain of a laminin-5α3 chain and the polypeptide specifically binds to at least one of α6 β1integrin receptor and α6 β4 integrin receptor that specifically binds toa polypeptide comprising a segment consisting of SEQ ID NO:6, and asecond component chemically bound to said first component.
 55. The fusedor chimeric polypeptide of claim 54, wherein the second component is apolypeptide.
 56. The therapeutic agent of claim 54 wherein the secondcomponent is a non-protein agent.